The present invention relates to a method of carrying out blood tests.
Many diverse blood tests are required for diagnosing, in particular, internal diseases and for controlling the course thereof. These blood tests can be divided into tests regarding the cellular blood components (blood count, differential blood count, blood group, immunophenotyping of blood cells) and into serum tests and also into antibody screening tests, Coombs tests and cross-matching tests. Serum tests are concerned with the determination of enzymes, metabolic products (e.g. creatinine, urea, blood sugar) and coagulation parameters. In addition, special tests, such as hormone analyses or drug level analyses, are carried out on the basis of serum.
With seriously ill and hospitalized grown-up patients, enlarged blood tests have to be carried out over a prolonged period of time every day and sometimes even twice a day to detect critical changes in the physical condition of the patient at an early stage. Blood is taken under sterile conditions normally using a closed blood taking system by puncturing a vein in the bend of the elbow (e.g. V. cubita mediana). Blood is optionally taken through already laid central indwelling catheters. Depending on the clinical requirements and the desired laboratory tests, types and numbers of the tubes to be filled with blood are defined. Specific laboratory tests can only be carried out with specific substances specifically prepared for such tests (e.g. specific anticoagulants (heparin, EDTA, citrate, etc.)) or with glucosidase inhibitor (xe2x80x9cblood sugar tubesxe2x80x9d) or blood withdrawing tubes containing coagulation-promoting substances (xe2x80x9cserum tubesxe2x80x9d). Heparin and EDTA are unspecific, indirect inhibitors. They do not act directly or specifically on a specific element of the blood coagulation cascade, but their effect is of an indirect nature in that they intercept, for example, Ca2+ ions which, in turn, are essential for the activation of different proteins of the coagulation system. A uniform pretreatment of the blood to be tested (xe2x80x9cstandard tubexe2x80x9d), on the basis of which all or at least the majority of important blood tests could then be carried out, does not exist. Furthermore, it follows from the standard logistic sequence in clinico-chemical or hematological laboratories that a plurality of blood withdrawal tubes that have been pretreated in the same way are often needed. After withdrawal from the patient, and depending on the laboratory values to be determined, the blood tubes are transferred into laboratories that are most of the time separated spatially (most of the time a clinico-chemical laboratory and a hematological laboratory and optionally laboratories for special tests, such as immunophenotyping, drug level in the serum, etc. (material dispatch by mail or courier might here be necessary). The blood tubes received in the laboratory must first be sorted, and the tubes to be centrifuged are then centrifuged at about 3,500 r.p.m. for five minutes. The blood taking tubes are then forwarded to the different work places (coagulation tubes to the coagulation place, serum tubes for electrolyte determination on the flame photometer, etc). The respective automatic analyzing devices are then loaded with the samples, with the sample volume of about 10 xcexcl to 100 xcexcl, which is needed for one measurement, being very small in most measuring operations. The actual measuring operation lasts from a few seconds to a few minutes (five minutes at the most, depending on the method and the device). The measured values are finally printed out and, depending on the origin, are communicated in writing to the dispatching stations.
In summary, it is necessary at the moment that several blood taking tubes (depending on the desired test) should be filled with 2-10 ml blood (depending on the tube size) in a blood taking process. The following tubes are needed for determining the routine laboratory parameters:
If, in addition, the blood group has to be determined and an antibody screening test has to be carried out and erythrocyte concentrates have to be provided for, two further blood taking tubes (without additions) have to be taken. In the case of special tests that are required, e.g. hormone level analyses (T3, T4, TSH basal, etc.), drug level (digitoxin level, vancomycin level, theophylline level, etc.), special electrolyte concentrations (magnesium, calcium, phosphate) and special coagulation values (deficiency in factors, fibrin degradation products), and many others, an additional withdrawal tube (most of the time serum tube) is needed for each test as a rule.
Hence, independently of the blood taking system, the following serious drawbacks are found in these blood taking methods that have so far been in general use:
1. The daily blood withdrawals which must be performed in the case of seriously ill patients lead to a blood loss of about 250 ml per week. This is an amount approximately half the blood donation amount of a healthy person at the German Red Cross. Another drawback is that seriously ill persons often suffer from anemia caused by very different factors.
2. The many blood withdrawing operations that are required are a considerable cost factor in medical care; on the one hand, because of the purchasing costs and, on the other hand, because of the considerable disposal costs for the tubes. The blood tubes used are classified as infectious wet waste and must be burnt being packed in special containers. In the case of seriously ill persons about 40 blood taking tubes are needed every week.
3. The workplace classification which is defined and segmented by the differently pretreated blood samples (anticoagulated whole blood for blood count determination, serum for enzyme tests and electrolyte determinations, etc.) and by the automatic measuring devices adapted thereto requires a multitude of work places entailing correspondingly high costs with respect to personnel and financing.
It is the object of the present invention to provide a method for carrying out blood tests, whereby the above-mentioned drawbacks can be overcome and many blood measuring parameters can be determined rapidly and reliably within a short time interval almost at the same time.
This object is achieved by a method according to claim 1. The achievement of such an object is, in particular, due to the finding that almost all of the clinico-chemical blood parameters can be determined using not only blood serum, but also blood plasma provided such blood plasmaxe2x80x94in contrast to standard practicexe2x80x94has been prepared by adding a thrombin inhibitor, such as hirudin. The difference between serum and plasma is that the first one is free from fibrin whereas the latter still contains fibrinogen. Surprisingly enough, it has been found that one and the same blood sample can be used for determining both clinico-chemical parameters and hematological parameters provided the sample is mixed with a thrombin inhibitor. Preferably, hirudin and/or desulfatohirudin is/are used as thrombin inhibitor. This method can be carried out in an automated manner.
Hirudin is a highly specific thrombin inhibitor which is naturally found in the salivary gland secretion of leeches, Hirudo medicinalis. The anticoagulative activity in salivary gland secretions of Hirudo medicinalis was described by Haycraft for the first time about 100 years ago (Haycraft, J. B. (1894), Naunyn-Schmiedebergs Arch. Exp. Pathol. Pharmakol. 18, 209). In the fifties Markwardt et al. succeeded in obtaining hirudin in pure form and in characterizing it biochemically (Markwardt, F. and Walsmann, P. (1958), Hoppe-Seyler""s Z. physiol. Chemie 312, 85). Hirudin is a polypeptide of which various, naturally occurring variants have become known in the meantime and which has a molecular weight of about 7000 Dalton. Natural hirudin is used as a thrombin inhibitor for biochemical studies (Walsmann et al., (1988) Pharmazie 43, 737). The use of hirudin has been limited to a very small number of very special applications because of the small hirudin amounts found in leeches and because of the troublesome extraction of hirudin. Among other things, hirudin has also been suggested for the anticoagulation of blood samples for determining the function and change in state of blood cells (e.g. blood sedimentation rate) (EP-A-442 843).
Since the end of the eighties it has been possible to prepare desulfatohirudin by genetic engineering techniques using yeasts or bacteria in great amounts. This recombinant desulfatohirudin is identical with the natural hirudin of Hirudo medicinalis (except for a missing sulfate group on tyrosine 63) and has the same anticoagulant characteristics. On account of the production costs, which are still high, recombinant hirudin is preferably developed for use in the therapeutic field. Its yield, however, could be increased by the use of novel expression systems, such as the yeast Hansenula polymorpha, to an extent (Weydemann et al. (1995), Appl. Microbiol. Biotechnol 44, 377) that non-therapeutical applications present themselves for hirudin. Recombinant desulfatohirudin is easily soluble and can already inhibit blood coagulation in small concentrations. This makes it possible to supply recombinant desulfatohirudin either in dissolved form or as a dry substance and thereby to effectively inhibit blood coagulation. The amount of the supplied desulfatohirudin can be dosed such that the blood taken remains incoagulable, depending on the time required by corresponding tests. Furthermore, the amount of desulfatohirudin can be proportioned such that there will be no dilution effects on the blood volume to be taken and the measurement result will thus not be influenced. The observation of standard volumes is a critical factor in the use of citrate solution as anticoagulant. In contrast to Na-EDTA or citrate solution, the anticoagulating effect in desulfatohirudin is not due to the withdrawal of Ca2+ ions, but to a specific steric thrombin inhibition. As a result, no bivalent cations are removed from the blood, which permits an examination of cellular blood components under physiological conditions. Moreover, desulfatohirudin does not require any endogenous factors for its anticoagulative activity, as is e.g. the case with heparins. For instance, heparin requires the factors antithrombin III and heparin cofactor 2 for efficiently inhibiting coagulation. With desulfatohirudin, blood samples that are derived from patients suffering from a corresponding factor deficiency can be analyzed without any additional measures and without any problems. It can be expected because of theoretical considerations that natural hirudin variants and hirudin fragments, as far as they have a thrombin-inhibiting effect, produce the same results as have been obtained for recombinant desulfatohirudin. Furthermore, synthetic thrombin inhibitors can be used with the same result.
When hematological parameters are examined, the blood cells must be vital at the time of examination. The blood must be anticoagulated and must not be changed in its volume parts by the course of the examination; otherwise, the numerical values would be distorted. Hematological test parameters are, e.g., the number of erythrocytes, leukocytes and thrombocytes per volume unit of whole blood, the proportionate composition of the leukocytes from the various nucleated blood cells which is determined under morphological criteria (differential blood), and the degree of loading of the individual erythrocytes with hemoglobin (quotient from the clinico-chemical measurement value hemoglobin concentration and the number of individual erythrocytes per volume unit). Further hematological measurement parameters using immunological test reactants describe the surface property (antigenity) of intact erythrocytes (transfusion-serological tests, cross-matching of conserved blood) or mononuclear blood cells (immunopnehotyping for diagnosing e.g. malign blood diseases). All of these hematological tests have in common that they can only be carried out on undestroyed, vital blood cells.
Hematological measurement parameters are determined either manually by microscope (e.g. by means of a counting chamber for determining the number of blood cells; blood smear preparation for assessing the cell morphology (cell size, nucleus shape, cytoplasma characteristics, cytoplasmatic granulation, etc.)) or in automatic blood-cell counting devices (e.g. Coulter counter). The determination of the hemoglobin value in blood is a clinico-chemical and not a hematological measurement parameter.
The present method is a method which can operate in an automated manner with whole human blood in a blood taking tube in a novel manner and in such an anticoagulated fashion that the blood sample can simultaneously be used for the automated determination of hematological, clinico-chemical and immunological measurement parameters, i.e. in short, for the determination of almost all measurement parameters. The routine measurement methods which have so far been in use can be employed entirely or after having been slightly adapted to the novel anticoagulant.
Furthermore, it should be noted that a quantitative determination by means of automatic measuring devices is possible according to the present method. Fresh capillary blood is used for a manual determination, e.g. in the counting of blood corpuscles. In large laboratories hematological tests, however, can no longer be carried out manually, but only by automated measuring devices. For this purpose venous K2-EDTA-anticoagulated whole blood is resorted to. Thanks to the addition of the anticoagulant dipotassium-ethylenediaminetetraacetic acid (K2-EDTA) in the worldwide standard concentration of 1 mg per 1 ml of blood, a high concentration of potassium ions is introduced into the blood sample, apart from the desired complexation of the calcium ions by the EDTA. Such an artificially high potassium concentration effects a change in the osmotic gradient for potassium between the leukocytes (granulocytes and lymphocytes) and the ambient aqueous environment, with the effect that the leukocytes lose cell water and shrink. After some time (30 minutes) a new osmotic gradient for potassium ions is obtained through compensation mechanisms of the leukocytes, and the cells recover part of the lost cell water. This new equilibrium remains stable for several hours. Of course, it is different in comparison with the native blood which has no K2-EDTA added thereto. It is only when the nucleated blood cells in K2-EDTA-anticoagulated blood have been adapted to this new equilibrium and have reached a shrunken, but stabilized form, that they can be counted in repeatable form in automated measuring devices and can reliably be distinguished from one another in volume, conductivity and stray-light characteristics (automated differential blood counting). An effect similar to the K2-EDTA sample pretreatment is not observed with hirudin, which seems to rule out the use of hirudin blood for automated measurements. Surprisingly enough, however, the present results show that quantitative hematological measurement parameters can be determined in automated measuring devices with hirudin-anticoagulated blood.
When hematological routine parameters, such as the number of erythrocytes, leukocytes and thrombocytes, are determined in an automated manner, the anticoagulants sodium citrate, sodium oxalate and heparin lead to wrong measurement results, which is generally known. The addition of sodium citrate or sodium oxalate to whole human blood will shrink the erythrocytes to such a considerable extent that the situation prevailing in the non-anticoagulated native blood cannot be inferred from the automated size determination of the erythrocytes and the calculated results, e.g. hematocrit. Moreover, when sodium citrate-anticoagulated blood is used, the numerical value of all of the blood cells measured and of the hemoglobin value must be corrected by the factor 1.1. What is even more disadvantageous is the fact that the numerical values of the blood cells vary at random, which could so far not be explained in a satisfactory manner, with the variations having a range of 10% in the case of thrombocytes and, in pathologically reduced thrombocyte values, even a greater range. When heparin-anticoagulated blood is used, unforeseeable, random spontaneous aggregations of thrombocytes and leukocytes may occur, so that during counting in automatic measuring devices partly falsely low measured values are determined for these cells. When anticoagulants, such as heparin, calcium citrate, but also K2-EDTA, are used, such measurement errors are above all observed in the determination of hematological parameters when during the automatic differentiation in the hydrodynamically focused sample flow, the leukocytes are differentiated according to volume (resistance measurement in direct current), conductivity (measurement of the internal conductivity of the cells with high-frequency alternating current) and stray-light characteristics (measurement of the typical surface structures of the cells and their peripheral granulation with a helium-neon laser) (automated differential blood counting). For instance, a heparin addition during automatic differential blood counting is, in particular, detrimental to an exact recognition of the basophilic granulocytes which are rather rare, but particularly important from a diagnostic point of view. Typically, an excessively high value of basophilic granulocytes is indicated. Moreover, such a wrongly determined blood count based on the heparin-anticoagulated whole blood is difficult to be checked manually, such a check, however, being imperative. On account of its great molecular electrostatic charge, the heparin addition interferes with the necessary stains (May-Grxc3xcnwald stain and Giemsa stain) with which the blood cells which have been smeared on an object carrier of glass are fixed and stained (panoptic staining according to Pappenheim). Thus, the addition of heparin effects a blue tinge of the nucleated blood cells, which makes it difficult or even impossible to distinguish the blood cells by microscope. Finally, serious measurement errors may even be caused by the K2-EDTA which is recommended and used worldwide for determining hematological routine parameters in automated measuring devices, but also in the case of manual determinations. It often happens that small blood clots are formed for the reason that the K2 EDTA-coated blood tubes are not immediately tilted and moved after the withdrawal of blood. Such partly clotted blood samples must not be processed, for they lead to wrong numerical values of the blood cells in the case of determining operations carried out by machine or manually. Moreover, they might clog the microcapillaries of the automatic measuring devices.
Since these many undesired interferences of the different substances for the anticoagulation of whole human blood have been known and could not be foreseen in detail upon the introduction of such substances, it could be assumed that hirudin-anticoagulated whole blood would also interfere in a disadvantageous manner with some of the important routine measurement methods. Therefore, there was some general prejudice among the experts that the determination of a multitude of clinico-chemical parameters and hematological parameters on the basis of a single blood withdrawal vessel is not possible.
With the method according to the invention, however, all of the relevant clinico-chemical and hematological values can be determined from a single blood withdrawal container at the same time. Possible blood value determinations comprise:
1. Determination of the serum parameters (e.g. alkaline phosphatase, amylase, cholinesterase, creatine kinase, GOT, GPT, xcex3-GT, HBDH, lactate, LDH, lipase, albumin, bilirubin, calcium, chloride, cholesterol, creatinine, iron, total protein, glucose, uric acid, urea, potassium, magnesium, sodium, triglycerides, C-reactive protein, immunoglobulins, transferrin, anti-streptolysin-O, rheumatoid factors, C3, C4, apolipoprotein, drug level). A determination is preferably carried out in automated measuring devices after separation of the corpuscular components from the whole blood. For a further simplification of the method the serum parameters can also be determined in automated measuring devices in which a separation of the corpuscular components is no longer required.
2. Examination of the blood count: partial blood count (automated) and differential blood count (manual and automated).
3. Determination of the blood group, carrying out antibody screening tests, carrying out Coombs tests and cross-matching erythrocytes concentrates.
4. Immunophenotyping of normal and malign mononuclear cells in blood and bone marrow.
A blood withdrawal tube (universal standard tube) which can be used for the present invention may be a blood withdrawal vessel that contains the thrombin inhibitor as a solution or as a dry substance or as a surface coating. The amount of the thrombin inhibitor should be proportioned such that for a period of time in which all of the above-described analyses can be carried out, the coagulation of the withdrawn blood volume is fully inhibited, thereby permitting a conduction of the measurements in the illustrated method without any problems. For instance, tubes, syringes, pipettes and capillaries of plastic material, glass or metal are suited as blood withdrawal vessels.
A further embodiment of the present invention is concerned with the performance of the blood test with automated measuring devices that combine the following functions in one unit:
determining all clinico-chemical parameters,
determining all hematological parameters,
wherein in the course of the measuring operation first the hematological values (blood count/differential blood count) should be recorded and then, following a selective removal of the cellular blood components (=hirudin plasma), the clinico-chemcial parameters (including special determinations) should be determined. The cellular components can e.g. be removed via a microfilter or by centrifugation. Furthermore, the above-described blood withdrawal vessels can also be used with measuring devices which carry out determining operations by means of test strips.
It is a further object of the present invention to provide a method of determining clinico-chemical blood parameters which can be carried out in a more simple and faster manner than known methods.
This object is achieved by a method set forth in claim 10. Surprisingly enough, it has been found that a freshly taken blood or bone marrow sample which is mixed with a thrombin inhibitor can be used for determining the above-mentioned clinico-chemical parameters. A separation of fibrinogen is not necessary. Preferably, however, the cellular and corpuscular components, as described above; can be separated from the blood plasma prepared in this manner.
It is advantageous to use hirudin and/or desulfatohirudin, preferably recombinant hirudin and/or desulfatohirudin, as the thrombin inhibitor.
In this method, too, the freshly taken blood sample should be put into a withdrawal vessel in which the thrombin inhibitor is provided.
The clinico-chemical parameters can be determined by means of an automated measuring device.